The detailed examinations of cell signaling pathways have become the focus of numerous research groups. Successful completion of such analyses will provide an immense amount of knowledge about the cellular processes and their misregulation, and offer numerous putative drug targets for many heretofore untreatable diseases. A vast majority of these signaling networks depend largely on protein phosphorylation, an essential post-translational modification that regulates numerous cellular functions, including cell cycle progression, proliferation, differentiation, signal transduction and apoptosis. Hunter, T. (2000) Signaling—2000 and beyond, Cell. 100, 113-127. It has been shown to be among the most widespread post-translational modifications known. In cells changes in phosphorylation dynamics within the cell have been linked to the onset and development of numerous diseases, most notably cancer. Blume-Jensen, P., and Hunter, T. (2001) Oncogenic kinase signalling, Nature. 411, 355-365. Consequently, detection of protein phosphorylation is very important for further understanding of an organism's signaling pathways to prevent such abnormalities.
Currently, one of the most common techniques used to analyze protein phosphorylation is through mass spectrometry. Aebersold, R., and Goodlett, D. R. (2001) Mass spectrometry in proteomics, Chem Rev. 101, 269-295. Mann, M., and Jensen, O. N. (2003) Proteomic analysis of post-translational modifications, Nat Biotechnol. 21, 255-261. This allows identification of novel phosphorylated proteins and even the sites of phosphorylation. Mass spectrometry, however, is biased toward some phosphorylated sites over others, thus complete analysis of the system is difficult. Furthermore, many groups do not have access to such an instrument due to high cost and the requirement for technical operating experience. The most commonly used methods in this category include the utilization of phospho-specific antibodies and 32P labeling in a Western blot or ELISA formats. Though there are phospho-specific antibodies available for purchase, to date, only general anti-phosphotyrosine antibodies are of high quality. Pandey, A., Podtelejnikov, A. V., Blagoev, B., Bustelo, X. R., Mann, M., and Lodish, H. F. (2000) Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors, Proc Natl Acad Sci USA. 97, 179-184. Phosphoserine and phosphothreonine residues detection is still sequence-dependant, resulting in partiality of these antibodies. As for the 32P labeling, although it has been vigorously employed for detection of phosphorylation experiments, the approach has a number of drawbacks, most familiar including working with dangerous radioactive materials and difficulty of identifying physiological phosphorylation. This arises from toxic effects 32P has on cells, inducing DNA fragmentation, changing cell morphology, causing cell cycle arrest and eventually resulting in apoptosis. Cooper, P. C., and Burgess, A. W. (1985) Biosynthetic labeling with 32P: radiation damage to mammalian cells, Analytical biochemistry. 144, 329-335. Hu, V. W, and Heikka, D. S. (2000) Radiolabeling revisited: metabolic labeling with (35)S-methionine inhibits cell cycle progression, proliferation, and survival, Faseb J. 14, 448-454. As a result, radioactive analyses of protein phosphorylation are more commonly carried out in vitro. To address the disadvantages of the current phosphoprotein detection methods, we introduce a novel soluble nanopolymer-based reagent for the detection of protein phosphorylation in a 96-well plate. Due to such drawbacks, there exists a need to simpler techniques that allows for the determination of protein of phosphorylation states. Some aspects of the instant invention seek to address this need.